Hepatitis C virus (HCV) infection is one of the major concerns in public health. Currently, optimal antiviral therapy with pegylated interferon-alpha plus ribavirin, cures ~50% of patients infected with HCV genotype 1 and ~80% of patients infected with HCV genotypes 2 and 3. One of the difficulties regarding our understanding on treatment resistance is related to the nature of current antiviral agents. Both interferon and ribavirin have long been known for their broad-spectrum antiviral activity by creating a non-specific antiviral status rather than the direct interaction with viruses. Consequently, no explicit targets on HCV genome have been documented. Studies on drug resistance with these agents have generated very controversial data through conventional approaches that frequently focus on short domains of the HCV rather than the entire viral genome. Using a novel long RT-PCR and cloning technology and well characterized serum samples from the Hepatitis C Antiviral Long-term Treatment against Cirrhosis trial (HALT-C), we propose a viral sequencing project through which viral mechanisms for the resistance to antiviral therapy will be exhaustively examined at multiple levels. HYPOTHESIS: HCV resistance to antiviral therapy is associated with region-dependent mutations of viral quasispecies at either single variants or the population level. Aim 1: To explore genetic signatures at both HCV isolate and quasispecies levels in null responders infected with HCV genotype 1a. The full-length HCV quasispecies profiles at the baseline will be generated from patients with either null or sustained virological responses (SVR), followed by comparative analyses to identify potential genetic signatures that are associated with the treatment resistance. Aim 2: To demonstrate if there are distinct quasispecies structures of HCV genotype 2 in terms of the high response rate to the antiviral therapy. The full-length HCV quasispecies profiles will be generated from twenty SVRs with HCV genotype 2a, followed by comparative analyses with those derived from HCV genotype 1a. Aim 3: To characterize mutational patterns associated with HCV re-emergence in patients with relapse after initial response to antiviral therapy. The relapse indicates the survival of HCV from a putative population bottleneck formed under antiviral therapy. How does HCV respond to such "in vivo" population bottlenecks? This issue will be addressed through a sequential comparative analysis of full-length HCV quasispecies profiles. Data from these studies will have immediate applications for rational design of future HCV antiviral therapy in which PegIFN-( and ribavirin are still the core components.